Biocompatible and Bioabsorbable Derivatized Chitosan Compositions

ABSTRACT

The invention relates to biocompatible, bioabsorbable derivatized non-crosslinked chitosan compositions optionally crosslinked to gelatin/collagen by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) for biomedical use and methods of making and testing such compositions, including a modified acute systemic toxicity test. The compositions comprise derivatized chitosan reacetylated to a degree of N-deacetylation (DDA) of between about 15% and 40%. The compositions are typically bioabsorbed in about 90 days or less and can be made to bioabsorb at differing rates of speed. The compositions are initially soluble in aqueous solution below pH 6.5. The compositions have an acid content that can be adjusted between about 0% (w/w) and about 8% (w/w) to customize the composition for uses that require and/or tolerate differing levels of cytotoxicity, adhesion, composition cohesion, and cell infiltration into the composition.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/211,632, filed on Mar. 14, 2014, which itself claims priority to U.S.provisional patent application Ser. No. 61/784,467, filed on Mar. 14,2013, the contents of which are fully incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract DAMD17-02-C-0095 awarded by U.S. Army Medical Materiel Development Activityand Contract N00014-10-M-0201 awarded by the U.S. Office of NavalResearch. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to biocompatible, bioabsorbablederivatized non-crosslinked chitosan compositions that may or may not becrosslinked to gelatin/collagen by1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) forbiomedical use and methods of making and testing such compositions. Thecompositions of the present invention comprise derivatized chitosanreacetylated to a degree of N-deacetylation (DDA) of between about 15%and 40%. As confirmed using a modified acute systemic toxicity testdeveloped by the inventors, the implantation of the present inventivecompositions into mammals does not produce toxic biodegradation speciesgiving rise to an elevated cytokine IL-1β response. The inventivecompositions are typically bioabsorbed in about 90 days or less and canbe made to bioabsorb at differing rates of speed. The inventivecompositions are initially soluble in aqueous solution below pH 6.5. Theinventive compositions have an acid content that can be adjusted betweenabout 0% (w/w) and about 8% (w/w) to customize the composition for usesthat require and/or tolerate differing levels of cytotoxicity, adhesion,composition cohesion, and cell infiltration into the composition.

BACKGROUND OF THE INVENTION

Chitin is a natural high molecular weight polymer widely found innature. It is the main component of insect and crustacean cuticle, andis also part of the cell walls of some fungi and other organisms. Chitinis generally extracted from its natural sources by treatments withstrong acid (to remove calcium deposits where required) and strongalkali (to remove proteinaceous residue). Chitin is insoluble undertypical aqueous conditions and is considered to be a relativelyintractable polymer (difficult to process). Dissolution of chitin toenable direct processing into fibers or other forms requires the use ofunattractive solvent systems that are generally corrosive and toxic.

Chitosan is produced at the industrial level by hydrolytic deacetylationof chitin. Chitin and chitosan are part of the glycosaminoglycan familyof polymers. Chitosan is typically derived from chitin by deacetylationin the presence of alkali. Chitosan is a generic term used to describelinear polysaccharides which are composed of glucosamine and N-acetylglucosamine residues joined by β-(1-4) glycosidic linkages (typicallythe number of glucosamines≧N-acetyl glucosamines) and whose compositionis soluble in dilute aqueous acid. The chitosan family encompassespoly-β-(1-4)-N-acetyl-glucosamine and poly-β-(1-4)-N-glucosamine withthe acetyl residue fraction and its motif decoration (either random orblock) affecting chitosan chemistry. The 2-carbon amino group on theglucosamine ring in chitosan allows for protonation, and hencesolubilization of chitosan in water (pKa≈6.5) (Roberts). This allows theready processing of chitosan into fibers, films, and other forms, aswell as the ability to prepare high purity chitosan for biomedical use.

Depending on original biological material sourcing and control ofprocessing of chitin to chitosan, poly-N-acetyl-glucosamine compoundsexhibit widely differing physical and chemical properties which areprocessable from aqueous solution. These differences are due tochitosan's varying molecular weights, varying degrees of acetylation,the presence of contaminants such as covalently bound, species-specificproteins, single amino acid and inorganic contaminants, etc.

Much attention has been paid to chitosan as a functional polymer becauseseveral distinctive biomedical properties such as non-toxicity,biocompatibility and biodegradability have been reported. Indeed,chitosan is widely regarded as being a non-toxic, biologicallycompatible polymer. (Kean, T. et al., 2005, Adv. Drug Deliv. Rev.62:3-11 (“Kean”); Ren, D. et al., 2005, Carbohydrate Res.340(15):2403-10 (“Ren”)).

The potential safe, biocompatible, and bioabsorbable use of chitosanmakes it an attractive natural material for use in biomedical implants.Further, deacetylated and partially deacetylated chitin preparationsexhibit potentially beneficial chemical properties, such as highreactivity, dense cationic charges, powerful metal chelating capacity,the ability to covalently attach proteins, and solubility in manyaqueous solvents. Also, it is conventionally understood that chitosanadheres to living tissue, acts as a haemostatic agent, promotes rapidhealing, and has antibacterial properties.

These chemical and biological properties are now beginning to proveuseful in many medical applications. Although chitosan compositions arebeing used increasingly in the United States, Europe, and Asia inexternal medical applications, such as wound composition products,sponges, powdered haemostatic agents, and antimicrobial gels,biocompatible and bioabsorbable chitosan compositions are yet to beapproved for internal surgical use.

Chitosan has not gained usage as a biocompatible and bioabsorbablebiomedical implant material, at least in part, because chitosancomprises a large group of structurally different chemical entities andits biodegradation properties are driven by multiple co-dependentfactors that render composition design unpredictable. Variousphysicochemical characteristics of chitosan, such as molecular weight,degree of deacetylation, and distribution of acetamide groups in thechitosan molecule, influence chitosan function and bioabsorbability.(Kofuji, K. et al., 2005, Eur. Polymer J. 41:2784-2791 (“Kofuji”)). Ofthese characteristics, molecular weight and the degree ofN-deacetylation (DDA) are believed to be the two most importantdeterminants of the bioabsorbability properties of chitosan. (Ren;Kean).

The in vivo degradation of chitosan is not fully understood but it isbelieved to occur by enzymatic cleavage of the polymer chain. (Kean;Ren). Lysozyme is the most prominent of the chitosan degrading enzymesin humans, however there are various other chitanases generally found inanimals, plants, and microbes. (Kean). The degradation behavior ofchitosan plays a crucial role in biocompatible material performance. Thedegradation kinetics may affect many cellular processes, including cellgrowth, tissue regeneration, and host response. (Ren). Investigationsregarding the degradation of chitosan by lysozyme indicate that the DDAof chitosan is one of the key factors controlling the degradation ofchitosan. (Id.). Also, it has been noted that N-substitution may affectenzymatic degradation. (Kean).

The rate of biodegradation and bioabsorption in vivo is also subject tothe competing process of foreign body encapsulation (fibrous capsuleformation) which may ultimately wall-off the bioabsorbing composition ifthe rate of its bioabsorption is sufficiently slow and the foreign bodyelicits a moderate inflammatory response to promote an enhanced rate ofencapsulation. Such encapsulation is undesireable for an intendedbioabsorbable composition since it can extend the residence time of thecomposition in vivo potentially from months to years. A reduced rate ofencapsulation combined with timely clearing and removal of the foreignbody is desired since protracted residence time can result in theadverse events of vascular and/or neural impingement as well as promoteinfection.

A prerequisite for effective scission of chitosan by lysozyme is thatthere are regular groupings of at least three consecutive N-acetylglucosamine monomers in the polysaccharide chain (Aiba), i.e., the DDAof chitosan is sufficiently low (<70% DDA) with the necessary N-acetylmotif structure to enable systematic enzymatic cleavage. Generally, themore acetyl groups on the chitosan, the faster its degradation rate.(Tomihata, K. et al., 1997, Biomaterials 18:567-575 (“Tomihata”)).

The water soluble range for chitosan above pH 6.5, which is between 45%and 55% DDA (Roberts 1992), often causes confusion in the determinationof absolute rates of scission and of bioabsorption since water solublechitosan will appear to bioabsorb more quickly when in fact it has onlydissolved. (Freier, T. et al., 2005 Biomaterials, 26 (29):5872-8(“Freier”)).

As a general matter in addition to its DDA, other chitosan moleculecharacteristics such as its molecular weight, viscosity, solubility, anddistribution of acetamide groups affect chitosan's bioabsorptionproperties.

Also, as indicated previously, biomaterial biocompatibility plays animportant role in bioabsorption. Biomaterials which are biodegradable byenzymatic, hydrolytic or oxidative pathways, but which only slightlyelevate the local biomaterial inflammatory response, will bioabsorb at aslower rate than biodegradable biomaterials that moderately elevate thesame response. Interestingly, chitosan at high DDA is shown to have verygood biocompatibility, with reported biocompatibility declining as DDAis reduced. (Tomihata).

It is conventionally understood that chitosan bioabsorption andbiodegradation requires chitosan with DDA less than 70% and more than40% DDA if the poly-β-(1-4) N-acetyl glucosamine is to still beconsidered chitosan (soluble in dilute aqueous solution). Pure chitin(DDA near 0.0) has shown to be bioabsorbable. (Tomihata). Chitosancompositions at about 70% DDA and higher demonstrate minimalbiodegradation due, at least in part, to lack of acetyl groups to promptenzymatic cleavage and/or lack of solubility. (See Freier, T. et al.,2005 Biomaterials, 26 (29):5872-8 (“Freier”)).

It has been found that within a week of implantation that these higherDDA chitosans, while showing very good biocompatibility, begin toexperience encapsulation. (Vandevord). As such, chitosan compositionsnear 70% DDA and higher, with their slow rate of biodegradation andencapsulation, may never fully resorb in vivo, and may produceundesirable encapsulation.

It is reported in the literature that chitosan having below a 70% DDA isdemonstrated as biocompatible and bioabsorbable and is proposed as safefor biomedical use. Only chitosan compositions having a DDA of betweenabout 40-70%, however, have been demonstrated to bioabsorb in vivo withthe definition of the poly-β-(1-4) N-acetyl glucosamine being chitin orchitosan at the lower DDA being dependent, as per Roberts, on its watersolubility at or below pH 6.5. The biocompatibility of thesebioresorbable chitosan compositions, although less than high DDAchitosan, has been reported to warrant further investigation. The numberof reported in vivo studies of bioabsorption of chitosan with actualbioabsorption occurring, however, is very low. (See e.g., Tomihata). Themajority of other studies purporting to study chitosan bioresorption useonly in vitro enzymatic conditions (generally lysozyme). As shown,lysozyme solution allows for analysis of the relative susceptibly ofchitosan to biodegrade in vivo, however, it cannot account forabsorption and biodegradation effects associated with biomaterialbiocompatibility and the biocompatibility of the biodegradationproducts.

The biocompatibility and bioabsorption of chitosan compositions withDDAs lower than 40% have not been widely investigated. This may be, inpart, due to the fact that achieving a chitosan with a lower DDA can bedifficult. (Ren). Also, lowering the DDA of chitosan below 40% toachieve faster rates of biodegradation is frustrated by the fact thatchitosan having a DDA less than 40% should make the chitosan insolublein aqueous solution below pH 6.5 and hence not chitosan as per theRoberts definition. (See e.g., Ren; Xu, J. et al., 1996, Macromolecules29:3436-3440 (“Xu”); Freier). Further, chitosans having DDAs below 50%are not typically commercially available.

Nonetheless, to the extent that lowering the DDA of chitosan maybeneficially serve to increase its rate of biodegradation, theconventional wisdom is that too fast a rate of biodegradation may beundesirable as it is well-known that the more rapidly biomaterialsbiodegrade, the more likely they are to elicite an acute inflammationreaction due to a significantly large production of low-molecular-weightcompounds within a short time. (Tomihata).

Additionally, preparing compositions to include chitosan having thewater soluble range of 45%-55% DDA will cause undesirable swelling andfluid absorption by the compositions that may cause undesirable andunpredictable fluctuations in implant size and performance duringbioabsorption.

As detailed below, the inventors of the present invention havesurprisingly discovered that, contrary to conventional wisdom andindustry practice, compositions comprising derivatized non-crosslinkedchitosan compositions with a DDA range between 40% and about 70% aretoxic when implanted, biodegraded, and bioabsorbed. The presentinventors have also surprisingly discovered that biocompatible,non-toxic and bioabsorbable biomedical chitosan compositions with a DDArange of between about 15% and 40% can be prepared that, upon implant,are at least 85% bioabsorbed within about 90 days or less. Accordingly,the present inventors have not only overcome widely held misconceptionsby those skilled in the art regarding the biocompatibility andbioabsorbability of compositions comprising chitosan with a DDA rangebetween 40% and about 70%, but they have achieved 1) the surprisingidentification of a biocompatible and bioabsorbable chitosan DDA rangeof between about 15% and 40%, 2) methods of making the inventivecompositions comprising derivatized non-crosslinked chitosan usingchitosan having a DDA range of between about 15% and 40%, and 3)developed a modified acute systemic toxicity test to ensure that thecompositions of the present invention, when implanted, do not producetoxic biodegradation species giving rise to an elevated IL-1β cytokineresponse.

BRIEF SUMMARY OF THE INVENTION

The present invention relates, first, to biomedical biocompatible andbioabsorbable compositions comprising derivatized non-crosslinkedchitosan with a DDA range between about 15% and 40% with the derivatizednon-crosslinked chitosan of that composition at least initially solublein aqueous solution at pH 6.5. The compositions of the present inventionare specifically intended for simple aqueous preparation ofbioabsorbable and biocompatible implantable constructs with completebiodegradation and bioabsorption within about 90 days or less. Byenabling ease of aqueous construct preparation (fiber, film,freeze-dried matrix, etc.) and acceptably rapid controlled bioabsorptionof a biocompatible material, the present invention reduces unnecessaryrisk to a recipient since prolonged presence of an inflammatory foreignbody or one that does not absorb can cause adhesion, encapsulation,and/or infection.

The present invention further relates to methods of makingbiocompatible, bioabsorbable compositions comprising derivatizednon-crosslinked chitosan (that may or may not be crosslinked toGelatin/Collagen by EDC) with a DDA range between about 15% and 40% forbiomedical use. In one embodiment, the compositions of the presentinvention are prepared by reacetlyation of chitosan that has beendeacetylated to a DDA of about 80% or higher in acetic acid to achieve aDDA range between about 25% and 40%. Specifically, reactivation from apure high DDA chitosan of between about 85% and 100% is preferred. Inaddition to direct reacetylation, the methods of the present inventionmay optionally include reduction of chitosan free amine functionality byreaction with an electrophile.

The inventive compositions are made so as to be at least initiallysoluble in aqueous solution below pH 6.5.

The term “chitosan” as used in the compositions of the present inventionrefers to chitosan that is, at least initially, soluble in an aqueoussolution having a pH below or at about 6.5. It is noted that thechitosan included in the compositions of the present invention may atsome point become insoluble, but that the insoluble material isnonetheless continuously referred to as chitosan throughout thisdisclosure based on its initial solubility in an aqueous solution havinga pH below or at about 6.5. Accordingly, disclosed herein are inventivecompositions which comprise chitosan and that may or may not, at somepoint, include an insoluble chitosan material.

The inventive compositions have an adjustable acid content of betweenabout 0% (w/w) and about 8% (w/w) which can be customized based on itsintended use and depending on requirements and/or tolerance of differinglevels of cytotoxicity, adhesion, composition cohesion, and cellinfiltration into the composition.

Additionally, the present invention further relates to methods oftesting such compositions. Specifically, the present inventors havedeveloped a modified Acute Systemic Toxicity (AST) Mouse Lesion Test(MLT) to ensure that the implantation of the present inventivecompositions into mammals does not produce toxic biodegradation speciesgiving rise to an elevated cytokine IL-1β response. The AST MLT involvesa partially lysozyme biodegraded chitosan that is predictive of chitosanbioabsorption toxicity.

The compositions of the present invention may be used in biomedicalapplications including, but not limited to, use in surgery, minimallyinvasive procedures (endoscopic and laparoscopic), hemostasis control,tissue filling, scaffolding, tissue regeneration, adhesion prevention,and drug delivery.

The compositions of the present invention may take several physicalforms including, but not limited to, sponges, membranes, scaffolds,films, gels, injectable gels and/or fluids, fibers, nanofibers, powders,etc.

The present inventive compositions overcome widely held and problematicmisconceptions regarding the safety and biocompatibility of compositionscomprising chitosan with a DDA of between 40% to about 70%, i.e., thatsuch compositions provide improved biocompatible and bioabsorbableimplant compositions. For the first time, the inventors havesurprisingly discovered safe, biocompatible, and bioabsorbablecompositions for implant comprising chitosan having a DDA range ofbetween about 15% to 40%. The inventors' identification of this lowerchitosan DDA range for biocompatible bioabsorbable internal use debunksnot only misconceptions regarding the safety of chitosan with a DDA ofbetween 40% to about 70%, but also works in opposition to various otherfactors known to those skilled in the art that have made attempts todevelop such compositions undesirable such as, solubility problems, lackof predictability, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate various exemplary embodiments.

FIGS. 1A and 1B. Slides showing histopathology of subcutaneous implantedmaterial (rat) seven days after implantation. FIG. 1A shows achitosan+20% acid (w/w) sample. FIG. 1B shows a chitosan with no acidsample.

FIG. 2. Graph showing in vitro simulated arterial wound sealing (SAWS)testing results for test samples with standard acid (˜20%) and low acid(˜3%) content.

FIG. 3. Graph showing lysozyme susceptibility of compositions comprisingchitosan having different DDA levels.

FIGS. 4A and 4B. Slides showing histopathology of an explanted testcomposition comprising chitosan having a ˜35% DDA after 8 days at 20×magnification (FIG. 4A) and at 60× magnification (FIG. 4B), respectively(rat, intraperitoneal).

FIGS. 5A and 5B. Slides showing histopathology of an explanted testcomposition comprising chitosan having a ˜35% DDA after 28 days at 20×magnification (FIG. 5A) and at 60× magnification (FIG. 5B), respectively(rat, intraperitoneal).

FIGS. 6A and 6B. Graphs showing extrapolated absorption times forcompositions comprising chitosan at 0% DDA (A) and ˜35% (B) DDA.

FIG. 7. Graph showing the total dermatitis score for individual rats andcorresponding rat sera IL-1β [pg/ml].

FIGS. 8A, 8B, 8C, and 8D. System of ranking extent of atopic dermatitislesions in rats and mice (images of lesions) following intraperitonealintroduction of bioabsorbed chitosan.

FIGS. 9A, 9 B, 9C, and 9D. Material Compositions and Key AlphaIdentifier Index.

FIG. 10. Rat Implantation Study Compositions With BiocompatibilityFollowing Bioabsorption.

FIG. 11. MLT Testing And Results Of Chitosan Material Compositions.

FIG. 12. Table of Hemostatic Efficacy Results.

FIG. 13. Bioabsorbability, Biocompatibility, And Hemostatic Efficacy.

DETAILED DESCRIPTION OF THE INVENTION

A. Compositions

Chitosan used in the present invention may be from any source.Industrial production of chitosan generally exploits crustacean shellwastes, for instance crab or shrimp shells. Alternative sources forchitosan include, for example, fungi and other industrial fermentationprocess by-products, such as the biomass collected after fungi or yeastfermentation.

The chitosan starting materials used in the present invention may behigh quality medical grade chitosans derived from shrimp and qualifiedaccording to ASTM F 2103-01. Preferably, the chitosan starting materialis at least about 75% to 100% deacetylated, more preferably the chitosanstarting material is at least about 85% to 100% deacetylated, and stillmore preferably the chitosan starting material is at least about 95% to100% deacetylated.

The chitosan starting material can be of pharmaceutical grade orequivalent quality, e.g., ultrapure FMC Novamatrix chitosan andacetylated ultrapure FMC Novamatrix chitosans. Advantageously, thechitosan may be pharmaceutical grade, prepared and processed accordingto GMP (good manufacturing practice) standards. The chitosan should notcontain excessive levels of heavy metals, proteins, endotoxins or otherpotentially toxic contaminants. The dry starting chitosan materialshould be at least 99% pure and demonstrate absence of heavy metals(Pb+Hg+Cd+As<500 ppb); undetectable residual protein (<200 ppm); and lowendotoxin (preferably <100 EU/g, more preferably <20 EU/g, and mostpreferably <5 EU/g).

The chitosan starting material used with the present invention shouldhave a weight average molecular weight of at least about 80 kDa, morepreferably, it has a weight average molecular weight of at least about250 kDa, and most preferably, it has a weight average molecular weightof at least about 150 kDa.

Preferably, the chitosan starting material has a viscosity (which isabout 100 centipoise to about 2,000 centipoise) at 25° C. in 1% w/wsolution in 1% w/w acetic acid (AA) with spindle LV1 at 30 rpm. Morepreferably, the chitosan has viscosity (which is about 125 centipoise toabout 1,000 centipoise) at 25° C. in 1% w/w solution in 1% w/w aceticacid (AA) with spindle LV1 at 30 rpm. Most preferably, the chitosan hasviscosity (which is about 150 centipoise to about 500 centipoise) at 25°C. in 1% solution w/w in 1% w/w acetic acid (AA) with spindle LV1 at 30rpm.

In the case of chitosan starting material: preferably, undissolved solidin a 1% w/w chitosan solution in 1% acetic acid at 25° C. is ≦10% w/w.More preferably, undissolved solid in a 1% w/w chitosan solution in 1%acetic acid at 25° C. is ≦2% w/w. Most preferably, undissolved solid ina 1% w/w chitosan solution in 1% acetic acid at 25° C. is ≦1% w/w.

In one embodiment, the deacetylated chitosan starting material isreacetylated to achieve the compositions of the present invention.Reacetylation of the deacetylated chitosan beneficially enhances controlof levels of deacetylation below 40% DDA, aids removal of undesirableresidual proteinaceous epitopes, and produces a more desirablecontrolled material. Reacetylation from a pure high DDA chitosan ofabout 85%-100% DDA is preferred since (1) the more robust deacetylationtreatment will more likely remove undesirable proteinaceous epitopes,(2) chitosan materials can be filtered since chitosan is soluble, (3)reacetylation of solubilized chitosan provides for more reproduciblerandom chitosan structure, and (4) the DDA range will be more uniform(less likelihood of presence of toxic biodegradation residue).

The reacetylated chitosan material used with the present inventionshould have a weight average molecular weight of at least about 90 kDa,more preferably, it has a weight average molecular weight of at leastabout 280 kDa, and most preferably, it has a weight average molecularweight of at least about 170 kDa.

Preferably, the reacetylated chitosan starting material has a viscosity(which is about 100 centipoise to about 2,000 centipoise) at 25° C. in1% w/w solution in 1% w/w acetic acid (AA) with spindle LV1 at 30 rpm.More preferably, the chitosan has viscosity (which is about 125centipoise to about 1,000 centipoise) at 25° C. in 1% w/w solution in 1%w/w acetic acid (AA) with spindle LV1 at 30 rpm. Most preferably, thechitosan has viscosity (which is about 150 centipoise to about 500centipoise) at 25° C. in 1% w/w solution in 1% w/w acetic acid (AA) withspindle LV1 at 30 rpm.

Purity of the reacetylated chitosan material is determined by infraredanalysis, preferably with demonstration of absence of the O-acylabsorption at 1740±10 cm⁻¹ and determination of residual moisture. Thepresence of small residual levels of o-acetylation should notsignificantly affect biocompatibility or efficacy. Its control providesa level of impurity control in the target derivatized chitosan forregulatory purposes.

The dried reacetylated chitosan can become insoluble if heated attemperatures above 40° C. and if stored for more than seven days at roomtemperature. Preferably the material is dried by lyophilization at lessthan 20° C. to near 5% w/w residual moisture. If more than seven days ofstorage is required, then preferably it should be stored at between 2°C.-8° C. or, more preferably, at −20° C. or below.

The following solubility profile is desired in the case of reacetylatedchitosan. Preferably, undissolved solid in a 1% w/w chitosan solution in1% acetic acid at 25° C. is ≦10% w/w. More preferably, undissolved solidin a 1% w/w chitosan solution in 1% acetic acid at 25° C. is ≦2% w/w.Most preferably, undissolved solid in a 1% w/w chitosan solution in 1%acetic acid at 25° C. is ≦1% w/w.

The reacetylated chitosan preferably has a DDA range of at least about15% to 40%, preferably about 15% to 35%, more preferably of at leastabout 20% to 35%, and most preferably at least about 20% to 30%.

Here, “DDA” or “degree of deacetylation” is defined to refer to thefinal proportion of amino groups in the 2-position of the D-glucosamine(D-glucopyranose) units constituting the chitosan (orpoly-β-(1-4)-N-acetyl-D-glucosamine orpoly-β-(1-4)-2-acetamido-2-deoxy-D-glucopyranose), which have beenconverted to free amino groups by deacetylation or any combination ofdeacetylation with reacetylation and/or substitution involving anelectrophile. 100% DDA chitosan (poly-β-(1-4)-D-glucosamine orpoly-β-(1-4)-2-amino-2-deoxy-D-glucopyranose) results from removal ofall acetyl (acetamido) functionality at the 2-position of theD-glucosamine.

Typically these electrophile/nucleophile (Lewis acid-base) reactionsprovide for amide formation, N-acylation, carboxylation and alkylationof the C-2 amine.

DDA according to the present invention is measured as determined by FTIRspectroscopic analysis. Baxter, A. et al., 1992, Int. J. Biol. Macromol.14:166-169 (“Baxter”); Miya, M. et al., 1980, J. Biol. Macromol.2(5):323-324 (“Miya”); Roberts, G. A. F., 1992, London: MacMillan 86-91(“Roberts”). This FTIR method provides an accuracy of DDA determinationof near ±3% DDA. Although the FTIR method was used predominantly in thisinvention, alternate techniques to determine DDA were used tocorroborate FTIR analyses. The other techniques used were ¹H NMR (ASTMF2260-03) and UV spectrophotometric determination of DDA usingconcentrated phosphoric acid as solvent (Hein 2008).

According to an embodiment, the bioabsorbable composition of the presentinvention, on wetting, has a pH that is compatible with internal use,preferably an initial pH between 5.5 and 7.5, which equilibrates within24 hours to physiologic pH near pH 7.4.

According to an embodiment, the composition is at least 85% bioabsorbedwithin about 100 days or around 14 weeks, of implant. In anotherembodiment, the composition is bioabsorbed within about 60 days, or nineweeks, of implant. In another embodiment, the composition is at least85% bioabsorbed within about 30 days, or four weeks, of implant. Inanother embodiment, the composition is at least 85% bioabsorbed withinabout 14 days, or two weeks, of implant. In another embodiment, thecomposition is at least 85% bioabsorbed within about seven days, or oneweek, of implant.

In an embodiment, the chitosan composition further comprises gelatin orcollagen. Use of high purity gelatin or collagen is a preferred agent tofoam the chitosan gel. Gelatin (porcine source) was obtained from Gelitaand contained endotoxin less than 90 EU/g. The gelatin had a 286 gbloom, 5.54 pH, 0.01% ash content, and less than 100 cfu/g bioburden. Ina preferred embodiment, a composition comprising chitosan and gelatin isfoamed and prepared in connection with lyophilization to make a lowdensity foam sponge. Gentle compression of this foam sponge produces ahighly compliant, adherent composition. Chitosan compositions in apreferred embodiment demonstrated acceptable levels of swelling inlength and width of not more than 20% on implantation. Depending on theoriginal sponge thickness dimension before thermal compression,thickness of the composition on implantation over 7-14 days may revertto the original sponge thickness. Implantation of compositions accordingto this preferred embodiment that contain residual acid in the range0-8% (w/w) resisted dissolution and were effective in controlling robustlevels of bleeding.

The compositions of the preset invention may further comprise additionalhydrophilic polymers and/or less hydrophilic polymers. The additionalpolymer may include, but is not limited to, collagen, collagenderivative, gelatin, alginate, chitosan, keratin, a hydrophilicpolyamine, a hydrophilic polyamine salt, polydiallyldimethylammoniumsalt, polyhexamethylene biguanide, polyaminopropyl biguanide, a chitosanderivative, polylysine, polyethylene imine, xanthan, carrageenan,quaternary ammonium polymer, chondroitin sulfate, a starch, a modifiedcellulosic polymer, a dextran, hyaluronan, carbopol, polyvinylpyrrolidone, hydrogenated vegetable oil, paraffin, polyethylene-oxide,polyvinylalcohol, polyvinylacetate, pullulan, pectin or combinationsthereof. The starch may include, but is not limited to, amylase,amylopectin and a combination of amylopectin and amylase. The modifiedcellulosic polymer may include, but is not limited to, ethylcellulose,methycellulose, hydroxypropylcellulose, hydroxypropylmethycellulose,hydroxyethycellulose, carboxymethylcellulose, oxidized cellulose orcombinations thereof.

The compositions of the present invention may further comprise an activeingredient. The active ingredient may include, but is not limited to,calcium, albumin, fibrinogen, thrombin, factor VIIa, factor XIII,thromboxane A2, prostaglandin-2a, activated Protein C, vitronectin,chrondroitin sulfate, heparan sulfate, keratan sulfate, glucosamine,heparin, decorin, biglycan, testican, fibromodulin, lumican, versican,neurocan, aggrecan, perlecan, lysozyme, lysly oxidase, glucose oxidase,hexose oxidase, cholesterol oxidase, galactose oxidase, pyranoseoxidase, choline oxidase, pyruvate oxidase, glycollate oxidase and/oraminoacid oxidase, D-glucose, hexose, cholesterol, D-galactose,pyranose, choline, pyruvate, glycollate, aminoacid, epidermal growthfactor, platelet derived growth factor, Von Willebrand factor, tumornecrosis factor (TNF), TNF-alpha, transforming growth factor (TGF),TGF-alpha, TGF-beta, insulin like growth factor, fibroblast growthfactor (FGF), keratinocyte growth factor, vascular endophelial growthfactor (VEGF), nerve growth factor, bone morphogenic protein (BMP),hepatoma derived growth factor (HDGF), interleukin, amphiregulin,retinoic acid, erythropoietin, mafenide acetate, silver sulfadiazine,silver nitrate, nanocrystalline silver, penicillin, ampicillin,methicillin, amoxicillin, clavamox, clavulanic acid, amoxicillin,aztreonam, imipenem, streptomycin, kanamycin, tobramycin, gentamicin,vancomycin, clindamycin, lincomycin, erythromycin, polymyxin,bacitracin, amphotericin, nystatin, rifampicin, tetracycline,doxycycline, chloramphenicol, cefuroxime, cefradine, flucloxacillin,floxacillin, dicloxacillin, potassium clavulanate, clotrimazole,cyclopiroxalomine, terbidifine, ketoconazole, paclitaxel, docetaxel,imatinib, exemestane, tamoxifen, vemurafenib, ipilimumab, dacarbazine,interleukin-2, abiraterone, doxorubicin, 5-fluorouracil, tamoxifen,octreotide, sorafenib, resveratrol, ketamine, diclofenac, ibuprofen,paracetamol, codeine, oxycodone, hydrocodone, dihydromorphine,pethidine, buprenorphine, tramadol, venlafaxine, flupirtine,carbamazepine, gabapentin, pregabalin, lidocaine, autologous cell lines,stem cells, and combinations thereof.

The present invention may be accomplished according to various methodsand the composition may comprise various forms, including but notlimited to freeze-dried sponge, tissue scaffolds, matrix, fiber, powder,sheet, film, membrane, nanofiber, nanoparticle and hydrogel. Thecompositions of the present invention may be deployed as eitherstandalone implantable rapidly absorbable devices or as rapidlyabsorbable surface coatings on implantable devices. In part due to theantibacterial nature of chitosan, implant of the absorbable compositionsof the present invention may beneficially also allow for the eliminationor reduction of ambient contamination that could cause secondaryinfection.

The rates and quality of biodegradation and bioabsorption of thecompositions of the present invention may be assessed using variousmethods. For example, the rate of bioabsorption may be determined usingvisual assessment and/or measurement regarding the amount of implantedmaterial that remains over time. Determination of the estimatedbioabsorption rate for a medical device for a particular applicationlocation in vivo requires implantation in that location of a similarsize test composition in thickness (and otherwise having uniformdimensions). Relative density or change in thickness cross-section isdetermined along the test composition in at least two animals per timepoint and across at least three time points in which percentage changeis more than 50%. Bioabsorption is considered achieved when 85% or moreof the test composition has been absorbed. This testing assumes that thetest composition generally degrades uniformly from the surface into thebulk and that it does not fragment into large (≧100 microns) pieces thatsubsequently migrate away from the implant site. Such fragmentationduring bioabsorption is undesirable. Testing also assumes that the lossof material is associated with biodegradation and is not by simpledissolution. There are a number of guidelines for performing in vivobiodegradation/bioabsorption studies: ASTM F2150-07 “Standard Guide forCharacterization and Testing of Biomaterial Scaffolds Used inTissue-Engineered Medical Products”, ASTM F1983 “Practice for Assessmentof Compatibility of Absorbable/Resorbable Biomaterials for ImplantApplications”, ISO 10993-9 “Biological Evaluation of Medical Devices—Pt9: Degradation of Materials Related to Biological Testing”. The in vivotesting used in the examples below for chitosan bioabsorption was guidedby the principles outlined in ASTM F2150-07, ASTM F1983 and ISO 10993-9.

Note that there is acceptance and guidance for standard hydrolytictesting in the case of biomaterials which biodegrade by hydrolysis: ASTMF1635 “Test Method for in vitro Degradation Testing of HydrolyticallyDegradable Polymer Resins and Fabricated Forms for Surgical Implants.”This is because hydrolysis is simple to model and can be readilycontrolled. In the case of those biomaterials that biodegradeenzymatically or by phagocytosis such as in the case of chitosan, invitro testing can provide a helpful guidance to what might be expected,however until in vivo studies for a particular implant location areperformed to corroborate in vitro testing results, the in vitro testresults cannot be considered reliable indicators of bioabsorption rate.

Non-toxicity according to the present invention may be assessed usingvarious standard methods during/after bioabsorption-biodegradation ofthe chitosan. Replication of bioabsorption-biodegradation chemicalchange in vivo is possible using lysozymal enzymatic pretreatment of thechitosan. Subsequent testing of the degraded chitosan in standardbiocompatibility assays such as the MEM elution test and/or the AcuteSystemic Toxicity test allow for rapid screening of toxicity. Asdescribed in section (C) below, the present inventors have developed amodified acute systemic toxicity test to detect an elevated cytokineIL-1β response after implantation of compositions prepared in accordancewith the present invention.

In a particularly preferred embodiment, the compositions of the presentinvention are in the form of a sponge that has been freeze-dried andcompressed.

In a particularly preferred embodiment, a freeze-dried sponge accordingto the present invention comprises derivatized chitosan with a DDA ofabout 15% to 40% and gelatin that are combined and foamed prior to beingfreeze-dried. The ratio of chitosan to gelatin in this inventiveembodiment may be any of 1:1, 2:1, 3:1, with a preferred ratio forhemostatic composition application being 3:1, a more preferred ratiobeing 2:1, and the most preferred ratio being 1:1.

The sponge embodiment of the present invention includes about 40% toabout 100% w/w, preferably about 45% to about 85% w/w, more preferablyabout 50% to about 75% w/w, even more preferably about 50% to about 65%,most preferably about 50% to about 55% w/w of the chitosan derivativematerial.

In an alternative embodiment, a foamed chitosan gelatin sponge maycomprise about 40% to about 100% w/w, preferably about 45% to about 85%w/w, more preferably about 50% to about 75% w/w, even more preferablyabout 50% to about 65%, most preferably about 50% to about 55% w/w ofthe chitosan derivative material and about 0% to about 60% w/w,preferably about 15% to about 55% w/w, more preferably about 25% toabout 50% w/w, even more preferably about 35% to about 50%, and mostpreferably about 45% to about 50% w/w of the gelatin material.

The inventive compositions may have an adjustable acid content ofbetween about 0% and about 8% which can be customized based on itsintended use depending on requirements and/or tolerance of differinglevels of cytotoxicity, adhesion, composition cohesion, and cellinfiltration into the composition. Preferred acids include acids suchas, acetic, carbonic, lactic, glycolic, citric, succinic, malic,glutamic, ascorbic, hydrochloric, malonic, glutaric, adipic, pimelic,tartaric, etc.

The inventive compositions have numerous biomedical applications,including, but not limited to, use in surgery, minimally invasiveprocedures (endoscopic and laparoscopic), hemostasis control, infectioncontrol, tissue filling, scaffolding, tissue engineering, tissueregeneration, controlled delivery of active agents, controlled drugdelivery and adhesion prevention and/or combinations of these.

B. Methods of Making Compositions According to the Present Invention

Various methods to achieve the production of chitosan having a DDA ofabout 15% to 40% are known to those of ordinary skill in the art.However, the inventors have advantageously developed a reacetylationchemistry technique that allows for a one step process to createchitosan having a DDA of between about 15% and 40%. When starting with ahigh purity chitosan form having a DDA of 75% to 100% and a purity of99% to 99.99999% this beneficially facilitates production ofbioabsorbable chitosan implant materials with controllable and specificdegradation profiles.

Accordingly, in a preferred embodiment, preparation of derivatizedchitosan involves a derivatization step of high purity chitosan with thestoichiometric addition (in slight excess) of acetic anhydride to a 2%solution of chitosan having a DDA of 80% or more in acetic acid. Afterreaction of the acetic anhydride with the chitosan to acetylate thechitosan to between about 15% to 40% DDA (as determined by FTIRspectroscopic analysis) the chitosan is raised to pH 13 by addition of10 M NaOH aqueous solution. This increase to high pH causes the chitosanto precipitate as a slurry and results in the hydrolysis of any chitosan0-acetyl esters. The NaOH and NaAcetate are subsequently removed bywashing or by dialysis with multiple changes of water until theconductivity of the chitosan slurry is near the conductivity of thewater (near 1 microSiemans/cm). The N-acetyl derivatized chitosan havinga DDA between about 15% to 40% may be resolubilized into aqueoussolution preferably by use of acetic acid. Once in aqueous solution, thechitosan may be processed into different physical forms. TheN-acetylation derivatization of chitosan as described previously is asubset of N-acylation of chitosan in which N-formyl, N-acetyl,N-chloroacetyl, N-ethyl, N-propyl, N-propionyl, N-isopropyl,N-(2-methylproprionyl), N-hydroxyethyl, N-succinyl, N-pentanoyl,N-carboxy, N-carboxymethyl, N-butyryl, N-(2,2-dimethylproprionyl),N-(3-methylbutyryl), N-(3,3-dimethylbutyryl), N-sulfonyl,N,N-dicarboxymethyl, N-butyl, N-pentyl and/or N-hexyl acyl modificationsmight be made to chitosan to achieve similar effect. Other genericchemical modifications involving nucleophile/electrophile reaction ofthe glucosamine C-2 nitrogen would include N-alkylation,N-alkylidene/N-arylidene derivitization, and metal chelation.

An alternate method of removal of excess acetic acid after thereacetylation step is to pour the reacetylated chitosan acid solution toa depth of 0.5″ in 1.2″ deep wells of Teflon™ coated aluminum molds,place the molds on flat stainless steel freeze drier shelf at −40° C.and freeze the solution before freeze-drying to sublimate all the freewater and acetic acid over a 48 hour drying cycle. This freeze-dryingpreparation of the acetylated chitosan provides for a sponge withresidual salt bound acetic acid that can be removed, if desired, by thevolatilization of acid under heat and humidity. This approachcircumvents the need to neutralize with base (NaOH or KOH) andsubsequently to remove the base by washing in large quantities of WFI(water for injection). A possible limitation of this approach is that if0-acetylation of the glucosamine/N-acetyl glucosamine has occurred (asevidenced by IR absorbance at 1740±10 cm-1) this may need to be treatedby base (NaOH or KOH) to remove the ester or else the presence of thisester would need to be controlled and the O-ester chitosan materialvalidated that it does not change the safety and efficacy profile of thechitosan.

In an alternative embodiment, the derivatization step involvesreacetylation of the chitosan having a DDA 80% or more by the methoddescribed above to at least as low as about 65% DDA, and then reducingthe percentage of chitosan free amine functionality even further tobetween about 15% and 40% by derivitization of the glucosamine C-2nitrogen with an electrophile. The N-acetylation derivitization ofchitosan as described previously is a subset of N-acylation of chitosanin which N-formyl, N-acetyl, N-chloroacetyl, N-ethyl, N-propyl,N-propionyl, N-isopropyl, N-(2-methylproprionyl), N-hydroxyethyl,N-succinyl, N-pentanoyl, N-carboxy, N-carboxymethyl, N-butyryl,N-(2,2-dimethylproprionyl), N-(3-methylbutyryl),N-(3,3-dimethylbutyryl), N-sulfonyl, N,N-dicarboxymethyl, N-butyl,N-pentyl and/or N-hexyl acyl modifications might be made to chitosan toachieve similar effect. Other generic chemical modifications involvingnucleophile/electrophile reaction of the glucosamine C-2 nitrogen wouldinclude N-alkylation, N-alkylidene/N-arylidene derivitization, and metalchelation. Generally, performing these reactions is within the knowledgeand skill of one skilled in the art.

Because acidic residue in an implantable material can be undesirable asit can lead to acidosis/cytotoxicity, use of acetic acid in casting orfreeze-drying of the chitosan compositions is preferred sincesubstantially all of the acetic acid can be removed from the finalcomposition by heating the composition from about 60° C. to about 130°C. Preferably, when removing the final fraction of acetic acid, thisheating process is performed with humidity controlled air of about 5% toabout 25%. Alternatively, where a certain amount of acid is desired inthe composition in order to affect its adhesion properties, acid may beleft within the composition in amounts ranging from about 2% w/w toabout 8% w/w.

The compositions of dried chitosan derivative compositions are preparedeither from a single aqueous acidic chitosan derivative solution or froma combination of an acidic chitosan derivative solution and an aqueousgelatin solution with or without the addition of water-soluble acid.These solutions may also include D-glucosamine, N-acetyl-D-glucosamine,sucrose, lactose, sorbitol, fructose, maltose, dextrose, glucose,polyethylene oxide and glycerol as plasticizers or rheology modifiers.These solutions may also include small amounts of active agents. Theacids used to dissolve the chitosan derivative and potentially includedas chitosan acid salts in the final compositions may include acids suchas acetic, carbonic, lactic, glycolic, hydrochloric, citric, andascorbic acids, etc. Other examples of acids that may be used includeformic (due to toxicity it would need to be fully removed byvolatilization), succinic, malic, glutamic, malonic, glutaric, adipic,pimelic, and tartaric acids.

Combined chitosan gelatin solutions are prepared by mass fractionalcombination of the prepared aqueous solutions of chitosan derivative andgelatin. In the case of gelatin and chitosan derivative solution, EDCmay be used in the gelatin chitosan mixture to covalently bind(crosslink) gelatin to chitosan. Foamed compositions of chitosanderivative and gelatin may be prepared by aeration whisking (filteredair of a flow-hood) of the combined gelatin chitosan mixture to apre-determined lower solution density (e.g., 1.0 g/cm³ to 0.6 g/cm³).Table 1 shows possible compositions of these solutions.

TABLE 1 Materials and Preferred Ranges Preferred range % Material Range% w/w w/w Chitosan/Chitosan 0.01-10   1-2   Derivative Gelatin  0-100-1   Acetic acid  0-80 1-4 (Higher amounts of acetic acid reduceschitosan viscosity) Carbonic acid 0-1 0-0.2 Lactic acid 0-5 0-3  Glycolic acid 0-5 0-3   Hydrochloric acid 0-2 0-0.5 Citric acid 0-10-0.1 Ascorbic Acid 0-8 0-2   Polyethylene oxide 0-3 0-0.1 Glycerol 0-30-0.1 D-glucosamine 0-1 0-0.1 Sucrose 0-1 0-0.1 EDC   0-0.1  0-0.01

The final solutions of chitosan derivative in the case of driedcompositions, were phase separated and dried. The preferred method ofphase separation was by freezing with introduction of a thermalgradient. Typically rectangular aluminum molds 28.5″×4″×1.05″ with toprecess of 28″×3.5″×0.8″ with a thin coating of Teflon™ were placedhorizontally on a flat surface and filled to a height of 0.4″ withchitosan derivative solution. The mold was then placed on a horizontalshelf which was either pre-cooled to near −45° C. or on the samehorizontal shelf near 25° C. which was then ramped at a cooling ratebetween 0.8° C./min and 2° C./min with final temperature near minus 45°C. (−45° C.). A delay interval of 5 minutes to 60 minutes may be used inthe case of the cooling ramp process. The purpose of the delay intervalis to moderate the thermal gradient through the 0.4″ layer of chitosanto control extent and location of ice nucleation. Alternatively, if thethermal gradient is insufficient to provide for ice nucleation thatfavors lamella phase separation structure in the frozen cake, thechitosan may initially be heated before loading in the mold or be heatedwithin the mold in the lyophilization to a temperature higher than 25°C. A preferred pre-cooling ramp solution temperature for improvedlamella structure is 45° C. A more preferred temperature is 30° C. Amost preferred temperature is 35° C. Typically the higher initialtemperatures without delay intervals are used in achieving desiredfreezing structure in high purity chitosan solutions with absence ofheterogeneous particles that would normally more readily promote icenucleation. The solution was left for between 60 to 180 minutes forfreezing to be completed. Typically this freezing was performed on acooling shelf inside the controlled environment of a freeze dryer (e.g.a Virtis™ 24 sqft freeze dryer).

Thermal gradients were achieved in the freezing solution by the directedrapid removal of heat at the mold base surface in direct contact withthe cooling shelf and delay in the removal of this heat caused by therelatively high heat capacity of the aqueous solution originally nearroom temperature, the depth of the same aqueous solution in the mold,and the thermal insulating properties of the solution as liquid and asice. Once the solution was adequately frozen, sublimation of the ice wasachieved by application of both heat and vacuum until residual moisturein the chitosan cake was less than 5% w/w. Typically with vacuum between100-300 milliTorr, the freeze dryer shelf temperature was graduallyincreased over 24 to 48 hours to near 25° C.

The increase in temperature was done in such a manner that the originalphase separated frozen sponge structure was preserved and there was nooccurrence of melt-back phenomena, that is, there was no loss ofstructure due to cake collapse because of ice becoming liquid. Onceachieving a shelf temperature of 25° C., a further 10 to 12 hours ofdrying was undertaken to ensure adequate uniform dryness of the sponge.

After freeze-drying, the dried sponges near 27″×3″×0.35″ were removedfor treatment to reduce the volatile acid fraction. If carbonic acid wasused to achieve solution and other acids were not present, then no extratreatment was required to remove the acid since the carbonic acid wasremoved in the freeze-drying step. In the case of other volatile acids,but those less volatile than carbonic acid, typically acetic and lacticacids, the heating of sponges containing these acids at near 80° C. (butalso at temperatures between 60° to 130° C.) in a humid environment (5%to 25% humidity) resulted in substantial removal of the volatile acidcomponent. For hemostatic applications, higher density of the originalfreeze dried sponges (near 0.03 g/cm³) is required to achieve suitablewet mechanical properties of the sponges. Typically this is performed byuniaxial thermal compression of the freeze-dried chitosan sponge atdensity near 0.03 g/cm³ to a density greater than 0.09 g/cm³ and greaterthan 0.25 g/cm³, but not greater than 0.5 g/cm³. Preferably, this isperformed by uniaxial thermal compression in the temperature rangebetween 60° C. to 85° C., preferably at 80° C., of the freeze-driedchitosan sponge at density near 0.03 g/cm³ to a density greater than0.07 g/cm³ and preferably greater than 0.12 g/cm³, but not greater than0.5 g/cm³.

As explained in the examples and in further detail below, the presentinventors have discovered that bioabsorption of chitosan compositiontest samples with a DDA higher than about 45% caused an undesirablelevel of cytokine response in test rodents which in some instances ledto skin lesions and/or systemic atopic dermatitis. The inventors wereable to monitor the level of cytokine response in animals receivingimplants by determining the presence of IL-1β cytokine in blood samples.The inventors also found that they were able to overcome elevatedcytokine response by preparing compositions comprising chitosan havingbetween about a 15% to 40% DDA.

In a particularly preferred embodiment, the inventors advantageouslyachieved a chitosan composition that is at least 85% bioabsorbed in 90days or less, demonstrated very good biocompatibility, and goodhemostatic efficacy in oozing surgical and high bleeding injuries andanti-coagulation. This preferred embodiment involves an acid massfraction of about 5% w/w or less and reacetylated chitosan with a DDA ofabout 40% or less, such as about 15% to about 30%.

C. Modified Acute Systemic Test

The standard acute toxicity test is mouse model (N=5) of systemictoxicity of a test material extract following intraperitoneal injection.The standard test method is described in ANSI/AAMI ISO 10993-11: 2006.Biological Evaluation of Medical Devices. Pt 11. Tests for SystemicToxicity. The test material extract is obtained by placing the testmaterial (˜0.8 g chitosan) of surface area 60 cm² in 20 ml of standardaqueous saline (9%) at 37° C. for 72 hours. The injected volume ofextract is 50 ml extract per kg of body weight (1.0 ml per 20 g mouse).

The modified acute systemic test system was developed as the result ofstandard biocompatibility testing being found to be non-predictive ofsevere systemic responses in certain low acid (<5% acetic w/w) chitosansafter 3-4 days intraperitoneal implantation (dose≈82 mg/kg, ˜14 mg in˜185 g animals) in a rat model. In fact standard (non-implant) ISO 10993biocompatibility testing, including the acute systemic toxicity test(AST) indicated that the chitosan materials that caused severe systemicreactions on implantation were biocompatible.

The systemic responses observed on implantation involved occurrence ofhair loss with skin lesions on tail, foot and face of the ratsconsistent with atopic dermatitis, as well as loss in appetite, lethargyand loss in weight. Typically the systemic response effects onimplantation were found to be present in animals in which chitosanbiodegradation occurred. The extent of the systemic response was foundto be the greatest in chitosans that biodegraded rapidly on implant. Thesystemic response was found to present on implantation in chitosans withat least a degree of deacetylation greater than 35% degree ofdeacetylation and at most a degree of deacetylation less than 70%. Thestrong systemic response was observed in chitosans with a degree ofdeacetylation close to 65%. Chitosans close to 65% degree ofdeacetylation, prepared either by direct deacetylation from chitin (0%degree of deacetylation) or by reacetylation from high purity chitosannear 90% degree of deacetylation, all showed the same severeinflammatory response when implanted. Chitosan near 65% degree ofdeacetylation that was partially crosslinked with gelatin through theglucosamine C-2 amine, although demonstrating biodegradation onimplantation, was not found to cause the atopic dermatitis response inrats, and the rats did not exhibit weight loss or lethargy. The samehigh degree of deacetylation chitosan that was reacetylated to 35%degree of deacetylation was found to remain soluble in dilute aceticacid solution (able still to be defined as chitosan) and was able to beprepared as a dressing form for use in the rat implantation study. Thislow degree of deacetylation (35%) was found to biodegrade rapidly onimplantation but not demonstrate any of the adverse systemic toxicityresponses found in the case of the higher degree of deacetylationchitosan that also degraded rapidly. Reacetylated chitin (near 0% degreeof deacetylation) also was prepared. This was insoluble in diluteaqueous acid solution. This reacetylated chitin also demonstratedbiodegradation and absence of adverse systemic toxicity.

Blood samples taken from the rats in the implantation study demonstratedthat the adverse systemic toxicity correlated with up-regulation of thecytokine IL-1r3 that is a member of the interleukin 1 cytokine family.This cytokine is produced by activated macrophages as a proprotein,which is proteolytically processed to its active form by caspase 1(CASP1/ICE). This cytokine is an important mediator of the inflammatoryresponse, and is involved in a variety of cellular activities, includingcell proliferation, differentiation, and apoptosis.

The modified AST test included an initial step of treating in lysozyme(0.37% w/w lysozyme solution at 37° C. for 24 hours) to prime the systemwith degradation products in the case of biodegradable chitosans. Inorder to dissolve chitosan in the AST elution fluid, residual aceticacid in the test material was generally high (>5% w/w). In order toensure detection of any systemic effect, high concentrations of testcompositions were used (dose≈2000 mg/kg, ˜40 mg per ˜20 g animal). Thenormal test window of 3 days for standard AST testing was extended to 7days for the modified test. Also, additional observational guidelineswere added that included monitoring the test animals for pilo-erectileresponses, presence of lesions, indications of hair loss, as well asstandard lethargy, appetite loss and weight loss. Also a ranking schemewas introduced to grade the severity of the lesions. In the case of themodified AST test, the control saline included 0.37% w/w lysozyme.Generally the modified AST test was run in combination with standardnon-degraded (non-lysozyme treated) samples under the same conditionsand over the same time period. This allowed for control of any toxicityeffect associated with residual acid in the sample.

The examples below are provided for disclosure only and are in no wayintended to be limiting on the invention as generally described herein.

Example 1 Reduced Acid Content and Cellular Infiltration in ChitosanCompositions

Low (5% w/w) to minimal (<1% w/w) residual acid salt content in acomposition of the present invention enhanced the composition'sbiocompatibility by removing composition dissolution effects andcytotoxicity directly associated with the chitosan acid salt. At lowerlevels (<3% w/w) of chitosan acid salt in the chitosan matrix, theoriginal structure of chitosan matrix within the composition ismaintained on wetting and viable cells readily infiltrate through thematrix. FIGS. 1A and 1B show two histopathology slides (rat, seven daysafter subcutaneous implant) that demonstrate the effect of the presenceof acid salt in test compositions on the ability of cells to infiltratethe composition.

The compositions shown in FIGS. 1A and 1B are chitosan (88% DDA nogelatin) compressed compositions. These compositions were prepared bydissolution of chitosan in aqueous acetic acid solution, pouring theaqueous chitosan acetic acid solution mixture into mold cavities;freezing and cryogenically inducing phase separation of ice and moldedchitosan acetate cake; sublimation of the ice; volatilization (or not)of acetic acid from the dried cake; and thermal compression of driedcake as described previously.

In FIG. 1A, with acid present (>5% w/w) the chitosan material of thecomposition was globular (loss of original matrix structure) and had nocellular infiltration, but in FIG. 1B when the acid was substantiallyremoved (<3% w/w) the chitosan matrix of the composition maintained itsoriginal lamella structure, appeared fibrillar, and shows considerablecellular infiltration. Acid was removed in the composition shown in FIG.1B by a volatilization process to less than 3% w/w.

This experiment demonstrates that a composition acid content greaterthan 5% w/w would adversely affect the bioabsorption rate of chitosanmaterials. The results of the study demonstrate loss of originalfreeze-dried internal interconnected lamella structure and adversecytotoxicity in the higher acid content chitosan matrices. Thiscombination of loss of interconnected porous structure and cytotoxicityseverely restricts the ability of cells such as neutrophils, leucocytesand monocytes to infiltrate the composition. As it is cellularinfiltration and phagocytosis which are primarily responsible forbiomaterial degradation processes, restricted cellular infiltrationinduced by water-soluble, cytotoxic chitosan acid salt adversely affectsbiodegradation and hence, rate of bioabsorption.

Additionally, the inventors have determined that higher weightpercentages of acid salt, such as greater than 8% w/w, may induceacidotic toxicity in the case of peritoneal implantation. Accordingly,the inventors determined that reducing the presence of acid salt inimplanted chitosan below 8% w/w facilitates chitosan absorption anddecreases the risk of acidotic toxicity.

The inventors also determined, however, that the removal or substantialreduction of acid from the compositions reduces tissue adherenceproperties. These altered adherence properties based on the amount ofacid present in the compositions can be used to customize thecompositions for various hemostatic applications, etc., to render thesecompositions more or less adherent and to take into account the toxicitytolerance of a contemplated application.

Example 2 Reduced Acid Content and Cellular Infiltration in 50:50Chitosan:Gelatin Compressed Compositions

Two different approaches were pursued in an effort to achieve increasedadherence efficacy of low-acid 50:50 chitosan:gelatin compressedcompositions that were either foamed or not foamed.

Both approaches removed acid by the volatilization process to a levelsufficient for achieving adherence (2%-5%) but also removed enough acidsuch that cytotoxicity or acidosis related toxicity were not majorconcerns.

The first approach started with a composition form having high adherenceproperties prior to low acid treatment with the goal of maintainingsufficient adherence properties to meet efficacy goals after low acidtreatment. The second approach involved a novel low acid productionprocess developed by the inventors to gently remove acid to minimize theeffects of moisture on the composition's micro-structure. This secondapproach used a low level of less-volatile co-acid (e.g. lactic,glycolic, malic, citric, succinic acids, etc.) which remained in thechitosan composition after the more volatile acid has been removedsubstantially by volatilization involving heat and humidity.

Interestingly, the reduction of acetic acid in the compositions to a lowcontent near 3% w/w resulted in enhanced adherence of the compressed50:50 chitosan:gelatin compositions when compared to higher acetic acidcontent compressed 50:50 chitosan:gelatin compositions (20% w/w).

FIG. 2 shows the results of simulated arterial wound sealing (SAWS) fortwo sets of paired compressed 50:50 chitosan:gelatin compositions. SAWSprovides the burst failure threshold that a standard HemCon compositioncan resist when attached to a standard flat PVC surface with stressapplied normal to the PVC surface through a central 4 mm diameter columnof bovine whole blood at room temperature under dynamic rampingconditions of 8 mmHg/s. Prior to the pressure ramp, there is apre-attachment 10 second submersion in bovine whole blood at roomtemperature and an immediate subsequent three minute pressure attachmentapplication of 55 kPa of the wetted composition to the PVC surface.

In FIG. 2, results from two identical pairs of compressed 50:50chitosan:gelatin compositions are shown with the exception that thefirst pair comprises foamed chitosan:gelatin compositions and the secondpair comprises chitosan:gelatin compositions that have not been foamed.The first pair of compressed 50:50 chitosan:gelatin foamed compositionsshowed that the low acid (˜3% w/w) composition had a failure pressure of1800 mmHg while the composition with an acid content close to 20% w/whad a failure pressure of 950 mmHg. The second pair of compressed 50:50chitosan:gelatin non-foamed compositions showed that the low acid (˜3%w/w) composition had a failure pressure of 1300 mmHg while thecomposition with an acid content close to 20% w/w had a failure pressureof 700 mmHg.

Thus, the inventors determined that the compressed 50:50chitosan:gelatin foamed compositions with a low (˜3% w/w) acid contentdemonstrated increased adherence when compared to non-foamed counterpartcompositions.

Example 3 Comparative In Vitro Bioabsorption Testing of 50:50Chitosan:Gelatin Foamed and Compressed Compositions Comprising Chitosanof Varying Degrees of Deacetylation

50:50 chitosan:gelatin foamed and compressed compositions, onlydifferent in degree of deacetylation (DDA), were tested in lysozyme at37° C. for time-dependent susceptibility to absorption. As shown in FIG.3, the compositions became increasingly susceptible to time dependentabsorption in lysozyme as the DDA decreased. The compositions wereprepared using the same procedures. The compositions were processed withPrimex chitosan (DDA≧80%), non-acetylated ultrapure FMC Novamatrixchitosan (DDA≧85%) and acetylated ultrapure FMC Novamatrix chitosans.Varying degrees of deacetylation, 71%, 63%, and 35% were obtained forthe test compositions.

The test compositions were suspended in a 0.37% w/w lysozyme solution at37° C. Visual inspection of the samples was made at regular intervals.Visual inspection was made daily during normal working day (8:00 am to5:00 pm) with at least one inspection at the beginning of the day andone at the end. The time of first appearance of degradation byappearance of sample fragmentation in the sample aqueous lysozymesuspensions was recorded as the initial time of degradation. The timethat suspended sample fragments no longer could be observed in the testmixture was recorded as time to 100% degradation.

Example 4 Rat, Intraperitoneal Bioabsorption Comparative Study ofVarious Compositions

A rat intraperitoneal absorption study was performed to determineabsorbability and biocompatibility of various compositions comprisingchitosan having a degree of deacetylation varying from 88% deacetylatedthrough to fully reacetylated chitosan (0% DDA).

a. Test Composition Components and Preparation

FIGS. 9A and 9B provide a Master Table of all sample and controlcompositions, treatments, dimensions, compressions and densities used inExamples 4 to 8. Samples A, B, C, E, F, G, H, I, J, K and M and controlsAC and AD were used in the rat implant study of Example 4.

Deacetylated chitosans having an 88% DDA and a 63% DDA were obtained asultrapure grade chitosan from FMC Novamatrix in Norway. The materialswere off-white powders, with less than 0.5% insolubles, less than 1% ashcontent, less than 0.3% protein content, less than 10 pm heavy metals,and less than 10 cfu/g bioburden. The molecular weight for the chitosanswas greater than 60 kDa for the number average. Endotoxin analysis wasperformed on the materials received and demonstrated endotoxin EU wasless than 10 EU/g. Water used in these studies was water for injection.Acetic acid was obtained from Emprove at 100% purity. The gelatin(porcine source) was obtained from Gelita and contained endotoxin lessthan 90 EU/g. The gelatin had a 286 g bloom, 5.54 pH, 0.01% ash content,and less than 100 cfu/g bioburden. The microdispersed oxidized cellulose(Na/Ca salt of polyanhydroglucuronic acid) was obtained from SynthesiaA.S. (Czech Republic) as a biomedical grade material. The crosslinkingagent 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)was obtained from Pierce, Cat no 22980. Acetic anhydride was of ACSreagent grade obtained from JT Baker. Infrared analyses of the finalreacetylated chitosan demonstrated that there was no residual 0-acetylester functionality (absorbance 1850 cm⁻¹).

All preparation of compositions was performed in a controlledenvironment that was monitored for environmental bioburden. Thecompositions were prepared from freezing and freeze-drying 2% w/waqueous solution of chitosan in 2% w/w aqueous acetic solutions or 1%w/w gelatin and 1% w/w chitosan aqueous solutions in 2% acetic acidaqueous solution. Test implant samples B, C, E, F, G, H, I, J, K, and Mwere close to 14 mg in mass while implant sample A with EDCcross-linking was 5 mg in mass. In the case of gelatin chitosancomposition (Sample A), EDC was added to a carbonic acid, acidifiedaqueous mixture (in the absence of other acid) to crosslink the matrix.Also another composition (Sample I) included microdispersed oxidizedcellulose (mdoc) in combination with chitosan. Two of the compositionswere prepared from chitosan that had been reacetylated from 88% degreeof deacetylation by application of acetic anhydride (Samples K and M).After freeze drying, the compositions with moisture content at or below5% w/w moisture were thermally compressed at 80±1° C. from close to 7 to8 mm thickness to either 2 mm or 1 mm thickness (only Sample A wasprepared at 2 mm thickness).

The eleven test compositions were investigated with controls of oxidizedcellulose matrix (Surgicel™) and sham surgery. The test compositionscomprised chitosan having degrees of deacetylation of about 88%, 71%,63%, 35% (reacetylated) and 0% (reacetylated).

b. Test Subjects

Rats (Rat, Sprague Dawley, male, 120-200 g, N=6 per condition and timepoint except sham where N=4) were anaesthetized, a sterile surgicalfield was prepared and a ventral incision was made to access theintraperitoneal cavity. A test composition piece (5 mm×10 mm×1 mm, 14mg) was attached by placement and holding over a laceration injury tothe liver for 2 minutes resulting in cessation of bleeding, and thesurgical site was closed. Generally chitosan was well adhered to theliver after being held for 2 minutes while the control Surgicel™ beingnon-adhesive only stayed in placed through clotting of blood around andthrough its non-woven structure. Animals were returned to their cages torecover from the anesthesia. Animals were provided water and food andmonitored for sign of loss of appetite or lethargy. Animals weresacrificed by administration of a lethal dose of barbiturate. Testsamples were blinded to surgical personnel and pathology until aftercompletion of the pathology report.

Initially explant times were designed to be 49 days and 97 days, howeverthis was changed to the shorter times of 7 and 28 days when unexpecteddermal lesions were exhibited early in the study. Blood specimens,target organs, dermal lesion biopsies (if present), and explanted testcomposition (if present) were collected from each test subject. Allblood samples were tested at time zero and at time of explant. At thetime of explant, subjects were inspected for general health and theintraperitoneal cavity was visually inspected for the test composition,test composition fragments, organ health, healing of the injured liverlobe, and surgical adhesions. Any fragmentation of the test compositionwas considered undesirable. The incidence of fragmentation and of dermallesions in the different test sample forms are listed in FIG. 10.

The dermal lesions (FIG. 10) were clearly visible on exposed skin aroundthe animal ears, nose, mouth, feet and tail. Once the subjects wereun-blinded, the lesions were found to be present only in the testsubjects of groups with 63% DDA chitosan. The lesions appearedapproximately three to four days post-implantation of the testcomposition on an injured liver in the rat intraperitoneal space. Thedermal lesions resolved after close to ten days following implantation.The bioabsorption study was terminated early to allow determination ofpossible causes of the dermal lesions. The conditions of samplepreparation, the blinding of the study, the handling of the samples andthe low residual acidity were sufficiently well controlled to eliminatepossibilities of contamination events and surgical procedural problems.The consistent appearance of the lesions in only the 63% DDA chitosangroups suggested a material inflammatory/toxicity problem.

The material compositions in which lesions were observed containedrapidly absorbing chitosan with a degree of deacetylation of 63% withunreacted C2 amine functionality (i.e., not crosslinked or reacted withan electrophilic species, etc.).

In terms of bioabsorption, absence of dermal lesions, absence of in vivofragmentation, ability to control bleeding (not accounted for in FIG.10) and excellent histopathology, the top performing sample was the50:50 chitosan:gelatin foamed and compressed composition (Sample K)prepared by reacetylation of chitosan from 88% DDA to ˜35% DDA chitosan.This composition did not give rise to dermal lesions or fragmentationand demonstrated substantial but incomplete absorption within 28 daysafter implantation. FIGS. 4A and 4B and 5A and 5B demonstrate desirableactive cellular infiltration into the 50:50 chitosan:gelatin foamed andcompressed composition Sample K at 8 and 28 days, respectively. FIGS. 6Aand 6B predict the time to complete absorption (to at least 85%absorbed) of test compositions Samples M and K respectively within 90days based upon measurement of the explanted material test compositionhistological cross-sectional area at 0, 8, and 28 days afterimplantation.

Example 5 Cytokine Elevation on Bioabsorption

To identify the source of the dermal lesions observed in the ratabsorption model as discussed above in Example 4, a number of cytokinepossibilities were considered. Cytokine IL-1β was chosen forinvestigation because it is a member of the interleukin 1 cytokinefamily. This cytokine is produced by activated macrophages as aproprotein, which is proteolytically processed to its active form bycaspase 1 (CASP VICE). This cytokine is an important mediator of theinflammatory response, and is involved in a variety of cellularactivities, including cell proliferation, differentiation, andapoptosis.

A study for elevation in IL-1β was conducted ex vivo using blood seracollected from the rat test subjects from Example 4 that demonstratedlesions, as well as, the control test subjects and the test subjectsthat did not demonstrate lesions. It was hypothesized that if the testcomposition was activating macrophages that are, in turn, producingIL-1β this may explain the lesions that present similar to atopicdermatitis. It was also hypothesized that the macrophage activation andsubsequent cytokine elevation are dependent upon the active breakdown ofchitosan material to its biodegradation products during thebioabsorption process.

The blood sera were tested using a standard ELISA (RandD Systems,Quantikine Rat IL-1/IL-1F2 Immunoassay, Cat No. RLB00). The sera samplesfrom the rats of Example 4 seven days after implantation were tested.The results of the blood sera testing demonstrated that rats withlesions exhibited a statistically significant (P<0.01) elevation ofIL-1β compared to rats with no lesions (see FIG. 7). The total lesionscale of 0-18 in FIG. 7 was based on the sum of a lesion ranking index(see FIGS. 8A, 8B, 8C, and 8D) of 0-4 being summed over the results from6 animals. The lesion scoring scale assigns lesion scores as follows:zero for no observed lesions; one for mild to red or crust; two formoderate to red with crusts; three for moderate or severe to ulceration;and four for severe to sloughing and necrosis.

FIG. 8A shows rat 90-14A with a tail lesion sated at three. FIG. 8Bshows an example of eyes rated at three and ears rated at two. FIG. 8Cshows a rat's nose and mouth rated at three. FIG. 8D shows a rat's noseand mouth rated at a three. Consistent with this finding, thecompositions comprising crosslinked chitosan did not exhibit elevatedIL-1β and the compositions comprising chitosan with DDA greater than 70%and less than 40% did not demonstrate elevated IL-1β while compositionscomprising chitosan materials with degree of deacetylation greater than35% but less than 70%, such as 63%, demonstrated elevated IL-1β as wellas atopic dermatitis.

The inventors found that the occurrence of dermal lesions is materialmediated and that the absorption process induces changes in the chitosanwhich mediates cytokine elevation. The inventors of the presentinvention also surprisingly and unexpectedly discovered thatcompositions comprising chitosan reacetylated below 40% DDA and,specifically, compositions comprising chitosan reacetylated to about 35%DDA bioabsorbs rapidly without causing up-regulation of IL-1β cytokine.

Example 6 A Rapid Screening Test for Bioabsorbable ChitosanBiocompatibility

It is evident that the intraperitoneal implantation of compositionscomprising non-crosslinked 63% DDA chitosan resulted in dermal lesionsin rats. Whether the 63% degree of deacetylation (DDA) chitosan wasproduced by directed deacetylation from chitin, or reacetylation from ahigh purity 88% DDA chitosan, compositions comprising either 65% DDAbioabsorbing chitosan produced elevated IL-1β and atopic dermatitis. TheIL-1β testing results of the rat sera supports the understanding thatthe degradation process or degradation products from compositionscomprising chitosan having about a 63% DDA activate the undesirablemacrophage production of IL-1β in the blood.

Standard biocompatibility testing for a rapidly bioabsorbable implantmaterial (limited implant contact duration) performed according to theInternational Organization for Standardization (Biological evaluation ofmedical devices—Pt 1 ISO 10993-1) suggests that chitosan having a 63%DDA has acceptable biocompatibility for implantation. The standard ISO10993-1 testing performed included tests for cytotoxicity, irritation,and acute systemic toxicity.

Importantly, however, because ISO 10993-1 standard testing was notpredictive of the dermal lesions observed during bioabsorption, thepresent inventors developed a modified Acute Systemic Toxicity (AST)test referred to as Mouse Lesion Test (MLT). Additionally, the inventorsdeveloped the MLT to provide a reliable test that does not requiresurgical implantation to screen for biocompatible bioabsorbablechitosan. This is because surgical implantation studies require highlyskilled personnel, special surgical facilities, and animal useapprovals, and can consume significant development resources.

The MLT test method was described previously in part C of this inventiondisclosure. The MLT is a modification of the standard ISO 10993-1 AcuteSystemic Test in mice. The differences between the MLT and AST in thetesting methodology are the following: (1) the MLT utilizes a higherconcentration of test composition than is specified for the standardAST; (2) the MLT test window is extended to 7-days from 3-days; (3)there are additional observational acceptance/failure criteria for thetesting lab other than weight loss and/or death; and (4) the testchitosan material is aseptically pre-degraded using lysozyme to primethe system with biodegradation products. The MLT is a low cost test thatcan be used as a standard test in contracting testing laboratories toquickly and effectively screen preparations of bioabsorbingpoly-β-(1-4)-N-acetyl-D-glucosamine (both chitin and chitosan) forundesirable IL-1β elevation associated with biodegradation productformation. As well as the standard toxicity acceptance/failure responsesof weight loss and/or death that are used in the AST test, the MLTincludes pilo-erection, lethargy, hair loss, and lesion appearance asthese are all manifestations of an elevated cytokine response. Thedosing regime in the MLT developed of 2,000 mg/kg (40 mg/20 g mouse) isconsistent with bioabsorption guidelines ASTM F2150-07, ASTM F1983 andISO 10993-9 and provides clear indication of presence or absence of theacute IL-1β response associated with bioabsorption of some chitosans.

Samples from the rat absorption study, including Sample C and Sample Kfrom Example 4, were tested using the MLT test (N=5). Sample C in whichlesions were observed (compressed compositions with <5% acid w/w contentcomprising wherein the 63% DDA chitosan was prepared using directdeacetylation) and Sample K in which no lesions were observed(compressed compositions with <5% acid w/w content comprising 50:50chitosan:gelatin foamed wherein the chitosan was prepared using 88% DDAchitosan reacetylated to about 35% DDA) were tested.

Discrimination was seen between test results for Sample C and Sample Kin mice. For test samples with Sample C pre-degraded with lysozyme, onday 4, all test mice scored 0 for lesions; however the laboratorytechnician noted that the animals' coats were rough in appearance. Theanimals were bright, alert, and responsive. On day 5, all of the testmice scored 1 (mild) for lesions. On day 6, all the test mice scored 2(moderate) for lesions. They were noted to have red, thickened, crustyskin all over their bodies. Their eyes were squinted and surroundingtissue appeared pink and swollen. They also appeared slightlydehydrated. The water bottle was checked and was functional. Theanimals' hair was noted to be patchy, especially on the abdomen. Fortest samples with Sample K pre-degraded with lysozyme, no lesionsappeared on test or control mice. All of the mice appeared normal andremained healthy thru to study conclusion (day 7).

Samples O, P, Q, R, S, T, U, and V comprised the same starting ultrapure88% DDA chitosan that was reacetylated to a final DDA with the onlydifference between these samples (other than the controls that were notpre-degraded with lysozyme) being extent of chitosan reacetylation fromaqueous acetic anhydride. All samples O, P, Q, R, S, T, U, and V werecompressed compositions with <5% acid w/w content comprising 50:50chitosan:gelatin foamed wherein the chitosan was prepared using 88% DDAchitosan reacetylated to the desired DDA.

Sample D was prepared using a compressed composition with <5% acid w/wcontent comprising 63% DDA chitosan prepared using direct deacetylation.

The composition of sample reference number N was prepared using UP FMCchitosan of 88% DDA as indicated in FIG. 9.

Sample AB was prepared from food grade glucosamine and was intended as acontrol for the monomer of poly-β-(1-4)-N-glucosamine

All samples in FIG. 10 were tested at 40 mg/ml. Injection of 1 ml of thesample pre-degraded extract was injected into the intraperitoneal cavityof a 20 g mouse to provide a 2,000 mg/kg dose. All testing was conductedfor a 7-day testing period with signs of toxicity being presented on day4 with highest levels being presented by day 7 with survival of theanimal. The results of this MLT testing are provided in FIG. 11.

As shown in FIG. 11, the MLT test demonstrated that compositionscomprising chitosan having a 35% DDA, or lower than 30% DDA, produced noclinical toxicity signs, such as pilo-erection, lethargy, weight loss,hair loss, lesion, and death. By contrast, compositions comprisingchitosan having a 45%, 60%, and 63% DDA produced clinical toxicitysigns, including one or more of pilo-erection, lethargy, weight loss,hair loss, lesion, and death. Accordingly, this test assistedidentification of the biocompatible DDA range chitosan of falling below45% DDA and encompassing 35% DDA and less than 30% DDA.

Example 7 Efficacy

Prototype chitosan hemostatic test compositions W, X, Y, and Z withcontrols AE and AC were evaluated for hemostatic efficacy in swinemodels of moderate and robust hemorrhagic bleeding.

The compressed compositions W, X, Y, and Z comprising 50:50gelatin:chitosan solutions described (see FIG. 9) used chitosan preparedwith ultrapure 88% DDA chitosan that was reacetylated to a desired DDAand also involving foaming the solution prior to freezing and freezedrying as described in Example 4. The solutions were foamed because theinventors found that foamed compositions enabled significant improvementin composition flexibility and tissue compliance which eased applicationof the compositions as well as enhanced hemostatic efficacy. Foaming ofthe 50:50 chitosan:gelatin solutions to near 0.6 g/cm³ density wasachieved by whisking the mixture to introduce aeration.

As shown in FIG. 12, the test compositions also employed different lowacid contents ranging between about 2.7% w/w to about 5% w/w.

The test compositions were evaluated for hemostatic efficacy in bothdifficult to control oozing surgical bleeding and high pressure highvolume traumatic injury-type bleeding.

Healthy swine of weight 80 lbs to 130 lbs were anaesthetized, alaparotomy was made to expose the abdominal cavity. Exposure wasperformed in a manner to avoid trauma to vascular, urinary, bilious, andlymphatic structures.

Swine parenchymal models of acute heparinized bleeding following eitherliver capsular stripping or spleen capsular stripping were employed asboth these models of parenchymal bleeding are typical of difficult tocontrol oozing surgical bleeding. A total of 17 swine were tested foreither or both of heparinized liver capsular stripping and spleenstripping.

For animals where both liver capsular stripping and spleen stripping,injuries were used: one wound was created on each of two liver lobes(left medial and left lateral) and one wound on spleen. An infusion ofheparin (10,000 u) was applied to maintain an activated clotting times(ACT) greater than 250 seconds. ACT was tested every 15 minutes andadditional heparin (10,000 u) was administered to maintain the ACT>250seconds. Hemostatic testing was performed with capsular strippinginjuries of 2 cm×2 cm×0.3 cm on the spleen and on the liver. Test andcontrol compositions were cut such that a 2 cm×2 cm piece would beinserted within the injury and a 5 cm×5 cm piece was placed over thisand over the injury. A successful test on the liver or the spleen wasone that after holding uniform pressure over the applied compositionswith 4″×4″, 48-ply surgical gauze for three minutes there was nosubsequent bleeding and there was no bleeding within 20 minutes ofremoval of pressure.

A total of 3 swine were tested using a swine aorta perforation model,which is a good model of severe hemorrhagic bleeding. These swine werenot heparinized. The abdominal aorta was exposed with placement of a ˜25cm diameter circular retractor inside the abdominal cavity immediatelyover the aorta. The injury to the aorta was made using a 3 mm scalpelincision through the aorta and removal of a contiguous 4 mm diametersection of the aorta using a 4 mm diameter vascular punch. Mean arterialpressure (MAP) was maintained near 60 mmHg using Hextend™ infusion. Testand control compositions were 5 cm×5 cm. An injury could be used up to 6times in an animal with MAP maintained at or near 60 mmHg and withremoval of a previous dressing and swabbing of the immediate wound areawith clean gauze wetted with saline to remove any chitosan residue. Asuccessful test on the aorta was one, that after holding uniformpressure over the applied composition with 4″×4″, 48 ply surgical gauzefor three minutes, there was no immediate bleeding and there was nobleeding within 30 minutes of removal of pressure.

During surgery it was observed that the low acid content compositionswhile generally less adherent to the tissue than compositions withhigher acid contents, had increased resistance to dissolution and stillexhibited very good efficacy. Also, the decreased adherence of the lowacid content compositions beneficially resulted in minimal tissue damageupon removal and eased residue clean up.

The test results in FIG. 12 show that the compositions comprisingchitosan having a DDA of 35% were effective in 67% of swine tested inthe heparinized liver capsular stripping model. The test results alsoshow that the compositions comprising chitosan having a DDA of less thanor equal to 30% were effective in 63% of swine tested in the heparinizedliver capsular stripping model, 80% of swine tested in the heparinizedspleen capsular stripping model, and 100% of swine tested in the swineaorta perforation model.

Example 8 Bioabsorption, Biocompatibility and Hemostasis of 35% DDAChitosan

Compressed compositions comprising 50:50 gelatin:chitosan foamedsolutions using chitosan prepared with ultrapure 88% DDA chitosan thatwas reacetylated to a 20% DDA and having an acid content close to orless than 5% were prepared as described in Example 4.

FIG. 13 shows the results of testing these compositions in 28-day ratintraperitoneal absorption, biocompatibility (MEM elution, AST, MLT,irritation) and swine hemostatic testing as described above in Example 7relative to a commercial hemostatic absorbable control composition,Surgicel™, composed of oxidized cellulose.

The tested composition which is about 30% bioabsorbed after 28 days ofimplantation, demonstrates good cytotoxicity, good acute systemictoxicity, acceptable low irritation score, and effective hemostaticcontrol in bleeding situations that are either difficult to controlsurgical-type bleeding or heavy pressure and heavy flow traumaticinjury-type bleeding. The tested composition meets all the requirementsof an absorbable composition and is shown to be superior to thecommercial control in terms of hemostatic efficacy.

1. A method of determining the biocompatibility of a biocompatible,bioabsorbable derivatized and reacetylated chitosan composition having adegree of N-deacetylation of between about 15% and 40% by measuringelicitation of an elevated cytokine IL-1β response using a modifiedacute toxicity test.
 2. The method of claim 1, comprising pre-degradingthe composition.
 3. The method of claim 1, comprising preparing a testextract by treating the composition in a lysozyme solution.
 4. Themethod of claim 3, wherein the composition is exposed to 0.37% w/wlysozyme solution at 37° C. for 24 hours.
 5. The method of claim 1,wherein the composition has a low acid content.
 6. The method of claim1, wherein the composition has an acid content of less than about 5%acetic acid.
 7. The method of claim 1, comprising dosing highconcentrations of test composition degradation product extract.
 8. Themethod of claim 7, wherein the dose has a concentration of about 2,000mg/kg or about 40 mg of test composition extract per about 20 g of testsubject weight.
 9. The method of claim 7, comprising ensuring detectionof systemic effect.
 10. The method of claim 1, comprising testing thecompositions in vivo for at least seven days.
 11. The method of claim 1,comprising monitoring the test animals for at least one of pilo-erectileresponses, presence of lesions, indications of hair loss, lethargy,appetite loss, and weight loss.
 12. The method of claim 1, comprisingusing a control saline including about 0.37% lysozyme.
 13. The method ofclaim 3, comprising running the modified acute toxicity test of lysozymetreated compositions together with non-lysozyme treated compositionsunder the same conditions and over the same time period to distinguishany toxicity effect associated with residual acid in the compositions.14. The method of claim 1, comprising avoiding the need for surgicalimplantation of the composition.
 15. The method of claim 1, comprisingtesting test subjects for the presence of elevated IL-1β in the blood.16. The method of claim 1, comprising screening compositionpreparations.
 17. A method of determining the biocompatibility of abiocompatible, bioabsorbable derivatized and reacetylated chitosancomposition by measuring elicitation of an elevated cytokine IL-1βresponse using a modified acute toxicity test.
 18. The method of claim17, comprising pre-degrading the composition.
 19. The method of claim17, wherein the composition has a low acid content.
 20. A method ofdetermining the biocompatibility of a biocompatible, bioabsorbablederivatized and reacetylated chitosan composition by measuringelicitation of an elevated cytokine IL-1β response using a modifiedacute toxicity test, comprising pre-degrading the composition, injectingpre-degraded composition derivatives into a test subject, and avoidingthe need for surgical implantation of the composition.